MiSeq Sample Sheet Quick Reference Guide ®

Transcription

MiSeq® Sample Sheet
Quick Reference Guide
FOR RESEARCH USE ONLY
Revision History
Introduction
Sample Sheet Parameters
Sample Sheet Settings
Adapter Settings
Naming the Sample Sheet
Technical Assistance
ILLUMINA PROPRIETARY
Part # 15028392 Rev. G
April 2013
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Part #
Revision
Date
15028392
G
April 2013
Updated the Adapter sample sheet setting to include the adapter
sequence for Nextera Mate Pair libraries.
Added the ReverseComplement sample sheet setting for Nextera
Mate Pair libraries using the Resequencing or Assembly
workflows.
15028392
F
March
2013
Updated the following sample sheet settings:
• CustomAmpliconAlignerMaxIndelSize—Changed default to 25
• FilterPCRDuplicates—Changed to FlagPCRDuplicates
• VariantCaller—Added Resequencing workflow
• EnrichmentMaxRegionStatisticsCount and
ExcludeRegionsManifestA—Added for the Enrichment
workflow
• Added VariantMinimumQualCutoff
• Removed VariantFilterQualityCutoff;
VariantMinimumGQCutoff is preferred
• Added requirements for Targeted RNA workflow
• Removed Index 2 as an option for Assembly and Small RNA
workflows
• Noted that custom indices can be used for the Custom
Amplicon workflow with MCS v2.2, or later
Removed custom primer instructions. See Using Custom Primers on
the MiSeq (part # 15041638).
15028392
E
November
2012
Added the following information:
• Description of Enrichment workflow, manifest, and data
section requirements
• Data section requirements for PCR Amplicon workflow
• Descriptions of sample sheet settings AdapterRead2 and
QualityScoreTrim
• Note about supported option for listing genome references for
multiple species in the same sample sheet when using MiSeq
Reporter v2.1
Updated the following information:
• Organized sample sheet settings into settings for sequencing,
analysis, and variant calling
• Updated Small RNA workflow to list the genome folder as
required in the sample sheet
• Updated sample sheet settings for variant calling to add
VariantMinimumGQCutoff, and to update StandBiasFilter and
MinumumCoverageDepth for the Enrichment workflow
15028392
D
July 2012
Added the following information:
• Added the PCR Amplicon analysis workflow for Nextera XT
libraries and information about the manifest file
• Noted that adapter trimming is recommended for longer read
lengths up to 250 cycles
• Added descriptions of sample sheet settings for
PercentTilesToScan and StrandBiasFilter
• Changed Setup Options screen to Run Options screen per MCS
v1.2
MiSeq Sample Sheet Quick Reference Guide
Description of Change
3
Revision History
Revision History
Part #
Revision
Date
15028392
C
April 2012
Updated the following information:
• Updated name of Amplicon workflow to Custom Amplicon
• Updated name of DenovoAssembly workflow to Assembly
• Added GenerateFASTQ workflow
• Listed genome folder as required for amplicon sequencing in
15028392
B
December
2011
Updated the steps in Setting Up the Sample Sheet.
Listed manifest files as required for TruSeq Custom Amplicon
libraries.
15028392
A
September
2011
Initial release
4
Description of Change
Part # 15028392 Rev. G
The sample sheet is a comma separated values (*.csv) file that stores information needed
to set up, perform, and analyze a sequencing run.
Illumina recommends that you create your sample sheet prior to preparing your sample
libraries. You can create your sample sheet using the Illumina Experiment Manager or
create it manually using a text editor, such as Excel or Notepad.
Before starting the run, make sure that the sample sheet is accessible to the instrument by
copying the sample sheet to a network location accessible from the instrument computer,
or copy the sample sheet from a USB flash drive to the instrument computer using the
Manage Files feature in MiSeq Control Software (MCS).
When the run begins, the software copies the sample sheet from the designated sample
sheet folder to the root of the MiSeqOutput folder, and then copies it to the root of the
MiSeqAnalysis folder. At the end of the run, the sample sheet is used for secondary
analysis by the MiSeq Reporter software.
Illumina Experiment Manager
The Illumina Experiment Manager is a wizard-based application that guides you
through the steps to create your sample sheet.
Using the Illumina Experiment Manager not only reduces syntax errors, but also
provides prompts for information that applies to your sample type and analysis
workflow. It provides a feature for recording parameters for the sample plate, such as
sample ID, project name, dual indices, and barcode information. The sample plate
information is imported Using the Illumina Experiment Manager, you can import
sample plate parameters to the sample sheet.
The Illumina Experiment Manager can be run on any Windows platform. To download
the software, go to the Illumina Experiment Manager support page on the Illumina
website (support.illumina.com/sequencing/sequencing_software/experiment_
manager/downloads.ilmn).
For more information, see the Illumina Experiment Manager User Guide (part # 15031335).
Sample Sheet Workflow
1
Create your sample sheet using one of the following methods:
• Illumina Experiment Manager—See the Illumina Experiment Manager User Guide,
Part # 15031335.
• Excel or Notepad—See Sample Sheet Parameters on page 6.
2
Name your sample sheet with the reagent cartridge barcode number associated with
the sequencing run, and use a *.csv extension. This enables the software to
automatically associate the sample sheet with the run. For more information, see
Naming the Sample Sheet on page 16.
3
Copy the sample sheet to the network location specified in Run Options in MCS, or
copy it to the instrument computer from a USB flash drive using the Manage Files
feature.
5
Introduction
Introduction
The sample sheet is organized by sections titled Header, Reads, Manifests, Data, and
Settings. Section headings are case-sensitive and shown in brackets [ ] in following
example:
Figure 1 Sample Sheet Example in Excel
Header Section
Parameter
Description
Investigator Name
Your name.
Project Name
Project name of your preference.
Experiment Name
Experiment name of your preference.
Date
Date of your experiment.
Workflow
Required
The workflow field must list the analysis workflow name
recognized by MiSeq Reporter. For more information, see the
Analysis Software Guide.
Assay
The name of the assay used to prepare your samples.
Chemistry
The recipe fragments used to build the run-specific recipe. If this
field is blank, the system uses the default recipe fragments.
Optional—For TruSeq RNA or TruSeq DNA libraries, leave this
field blank.
Required—For any workflows that use dual indexing, specifically
Nextera and TruSeq Custom Amplicon, the chemistry field is
required. Enter amplicon in this field.
Reads Section
6
Parameter
Description
Number of cycles
in Read 1
Required
Number of cycles
in Read 2
Required for paired-end runs.
Part # 15028392 Rev. G
NOTE
The number of cycles for the index read is indicated by the index sequence defined
in the Data section.
Data Section
The following table summarizes the Data section requirements for each analysis
workflow. Column order is not important.
Workflow
Required Data Columns
Optional Data Columns
Assembly
SampleID
Sample_Name, Index,
GenomeFolder
Custom Amplicon
SampleID, Manifest, GenomeFolder,
Index, Index 2*
Sample_Name
Enrichment
SampleID, Manifest, GenomeFolder
Sample_Name, Index,
Index 2
GenerateFASTQ
SampleID
Sample_Name, Index,
Index 2
LibraryQC
SampleID, GenomeFolder
Sample_Name, Index,
Index 2
Metagenomics
SampleID
Sample_Name, Index,
Index 2
PCR Amplicon
SampleID, Manifest, GenomeFolder
Sample_Name, Index,
Index 2
Resequencing
SampleID, GenomeFolder
Sample_Name, Index,
Index 2
SmallRNA
SampleID, GenomeFolder,
Contaminants, miRNA, RNA
Sample_Name, Index
Targeted RNA
SampleID, Manifest, GenomeFolder,
Index, Index 2
Sample_Name
* For Custom Amplicon—Using MCS v2.2, or later, you can optionally specify a single index run and
use custom indices. The length of the index is read from the index sequence written in the sample
sheet. However, you must use the adapter supplied with Illumina kits.
Column
Heading
Description
SampleID
Required
Every sample must have a unique sample ID.
At least one sample must be listed. List one sample per line.
Sample_Name
Optional
The sample name is used in reporting and file naming.
Index
Required for multi-sample assays with single or dual indexing.
Nucleotide sequence—Valid characters are A, C, G, T, and N, where
N matches any base. Enter the index sequence of the i7 index.
7
Column
Heading
Description
Index2
Required for multi-sample assays with dual indexing.
Nucleotide sequence—Valid characters are A, C, G, T, and N, where
N matches any base. Enter the index sequence of the i5 index.
NOTE
For the appropriate index sequences, see the user guide for your sample preparation kit.
Genome Folder Path
The GenomeFolder is the folder containing the reference genome in the FASTA file
format, which can reside on either a network location or the MiSeq local drive. For
optimal results, store reference genomes on the local drive or use BaseSpace.
Enter the full path (UNC path) to the GenomeFolder in the sample sheet. Do not enter the
path using a mapped drive.
NOTE
Introduced in MiSeq Reporter v2.1, you can specify genome references for
multiple species in the same sample sheet for all workflows except the Small RNA
workflow.
Data Section Requirements by Workflow
Not all columns used in the Data section of the sample sheet apply to every analysis
workflow. For example, the Manifests column is required for some workflows, and not
required for other workflows.
Data Columns for the Assembly Workflow
Column Heading
Description
GenomeFolder
Optional If provided, MiSeq Reporter compares the de novo
assembly against the reference genome, and generates a dot-plot
that graphically summarizing the results.
• If the specified folder does not exist, Illumina Experiment
Manager combines the GenomePath configuration setting with
the genome string.
• If the path does not exist, MiSeq Reporter stops processing.
Data Columns for the Custom Amplicon Workflow
8
Column Heading
Description
GenomeFolder
Required The reference genome folder containing the FASTA files
to be used in the alignment step.
The reference genome must be the same genome used to generate
the manifest file. The genome folder is used to provide variant
annotations and set the chromosome sizes in the BAM file output.
Manifest
Required Specify the manifest key for this sample, which is located
in the first column of the Manifests section.
Part # 15028392 Rev. G
Column Heading
Description
GenomeFolder
Manifest
Data Columns for the Library QC Workflow
Column Heading
Description
GenomeFolder
Data Columns for the PCR Amplicon Workflow
Column Heading
Description
GenomeFolder
Manifest
Data Columns for the Resequencing Workflow
Column Heading
Description
GenomeFolder
Data Columns for the Small RNA Workflow
Column Heading
Description
GenomeFolder
Optional If provided, reads are aligned against the full reference
genome.
Contaminants
Required The path to the folder containing the FASTA files of
contaminants.
miRNA
Required The path to the folder containing FASTA files of mature
miRNAs.
RNA
Required The path to the folder containing FASTA files of small
RNAs.
9
Data Columns for the Enrichment Workflow
Data Columns for the Targeted RNA Workflow
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Column Heading
Description
GenomeFolder
Manifest
in the first column of the Manifests section of the sample sheet.
Part # 15028392 Rev. G
The Settings section of the sample sheet is optional for all workflows. Settings control
various sequencing and analysis parameters. Each line in the Settings section contains a
setting name in the first column and a value in the second column.
Sample Sheet Settings for Sequencing
Parameter
Description
CustomRead1PrimerMix
CustomIndexPrimerMix
CustomRead2PrimerMix
Create one line for each custom primer used and indicate C1 for
the Read 1 primer, C2 for the Index primer, or C3 for the Read 2
primer. Custom primers are supported for Read 1, Index 1 Read,
and Read 2 only.
For more information, see Using Custom Primers on the MiSeq (part
# 15041638).
PercentTilesToScan
If set to the default value of 1, 100% of the tiles are scanned. Valid
values are 0 through 1.
• If set to 0, the software will round up to one tile.
• For all other settings, the software will round down. For
example, a value of 0.99 results in 27 tiles (dual-surface) or 13
tiles (single-surface).
For more information about dual-surface scanning, see the MiSeq
System User Guide, Part # 15027617.
Sample Sheet Settings for Analysis
Parameter
Description
Adapter
Specify the 5' portion of the adapter sequence to
prevent reporting sequence beyond the sample
DNA.
• Nextera libraries—Illumina recommends adapter
trimming for Nextera libraries (adapter sequence
CTGTCTCTTATACACATCT).
• Nextera Mate Pair libraries—Illumina
recommends multiple adapter trimming for
Nextera Mate Pair Libraries (adapter sequences
CTGTCTCTTATACACATCT and
AGATGTGTATAAGAGACAG).
To trim two or more adapters, separate the
sequences by a plus (+) sign (e.g.,
CTGTCTCTTATACACATCT+
AGATGTGTATAAGAGACAG).
• Small RNA libraries—By default, adapter
trimming is performed using the standard adapter
sequence TGGAATTCTCGGGTGCCAAGGC.
This adapter can be overridden using the sample
sheet.
For other libraries, see the associated sample prep
documentation.
11
Parameter
Description
AdapterRead2
Specify the 5' portion of the Read 2 adapter sequence
to prevent reporting sequence beyond the sample
DNA. Use this setting to specify a different adapter
other than the one specified in the Adapter setting.
Aligner
For Resequencing and Library QC workflows
As of MiSeq Reporter v2.2, Eland has been
deprecated, but not removed. You can use the
Aligner setting to specify Eland, if needed for
backward-compatibility.
When using the default aligner for any workflow,
you do not need to specify the alignment method in
the sample sheet.
CustomAmpliconAlignerMaxIndelSize
12
For Custom Amplicon workflow
By default, the maximum detectable indel size is 25.
(missing snippet link)
EnrichmentMaxRegionStatisticsCount
For the Enrichment workflow
Default is 40000. Sets the maximum number of rows
shown in the Targets table and recorded
EnrichmentStatistics.xml.
ExcludeRegionsManifestA
For the Enrichment workflow
Use this setting to exclude one of more region
groups (separated by plus signs) from consideration.
For example, if this is set to ABC+DEF, any region
that has either ABC or DEF specified in the Group
column of the manifest will be ignored when parsing
the manifest. This means that no variant calling will
be performed for this region and it will not be
reported in enrichment statistics.
If the sample sheet contains more than one manifest,
use multiple lines, such as ExcludeRegionsManifestB,
ExcludeRegionsManifestC, etc.
FlagPCRDuplicates
Settings are 0 or 1. Default is 1, filtering.
If set to 1, PCR duplicates are flagged in the BAM
files and not used for variant calling. PCR duplicates
are defined as two clusters from a paired-end run
where both clusters have the exact same alignment
positions for each read.
(Formerly FilterPCRDuplicates. FilterPCRDuplicates
is acceptable for backward compatibility.)
Kmer
For the Assembly workflow
Use this setting to override the k-mer size used by
Velvet. Default is 31; odd-numbered values up to 255
are supported.
QualityScoreTrim
If set to a value > 0, then the 3' ends of non-indexed
reads with low quality scores are trimmed.
Trimming is automatically applied by default at a
value of 15 when using BWA for alignment.
Part # 15028392 Rev. G
Description
ReverseComplement
Settings are 0 or 1. Default is 1, reads are reverse
complemented.
If set to true (1), all reads are reverse complemented
as they are written to FASTQ files. This is necessary
only in certain cases, such as processing of mate pair
data using BWA, which expects paired-end data.
Note that per-cycle metrics might be disrupted by
this setting.
Set this setting to 1 when using the Resequencing or
Assembly workflows with Nextera Mate Pair
libraries.
TaxonomyFile
For the Metagenomics workflow
Use this setting to override the taxonomy database;
default is taxonomy.dat.
VariantCaller
For the Custom Amplicon and Resequencing
workflows
Specify one of the following variant caller settings:
• GATK (default)
• Somatic (for tumor samples)
• Starling (legacy variant caller)
• None (no variant calling)
When using the default variant caller for any
workflow, it is not necessary to specify the variant
calling method in the sample sheet.
Sample Sheet Settings for Variant Calling
Setting Name
Description
FilterOutSingleStrandVariants
This setting filters variants if they are only found in
one read-direction.
This setting applies to the Resequencing and PCR
Amplicon workflows only; it does not apply to the
Custom Amplicon workflow.
Default value and variant caller:
• 1 (on)—Somatic Variant Caller (0 for Custom
Amplicon workflow)
IndelRepeatFilterCutoff
This setting filters indels if the reference has a 1-base
or 2-base motif over 8 times (by default) next to the
variant.
• 8—Somatic Variant Caller
• 8—GATK
• 8—Starling
13
Parameter
14
Setting Name
Description
MinimumCoverageDepth
The variant caller filters variants if the coverage depth
at that location is less than the specified threshold.
Decreasing this value will increase variant calling
sensitivity, but raise the risk of false positives.
• 20—GATK (Enrichment workflow only; 0 for any
other workflow)
MinQScore
This setting specifies the minimum base call Q-score to
use as input to variant calling.
• 0—Starling
StrandBiasFilter
This setting filters variants with a significant bias in
read-direction. Variants filtered out in this way will
have sb in the filter column of the VCF file, instead of
PASS.
• 0.5—Somatic Variant Caller
• -10—GATK (Enrichment workflow only; no filter
for any other workflow)
VariantFrequencyEmitCutoff
This variant caller does not report variants with a
frequency less than the specified threshold.
VariantFrequencyFilterCutoff
This setting filters variants with a frequency less than
the specified threshold.
• 0.20—GATK
• 0.20—Starling
VariantMinimumGQCutoff
This setting filters variants if the genotype quality
(GQ) is less than the threshold. GQ is a measure of the
quality of the genotype call and has a maximum value
of 99.
(VariantFilterQualityCutoff is acceptable for backward
compatibility.)
• 30—GATK
• 20—Starling
VariantMinimumQualCutoff
This setting filters variants if the quality (QUAL) is less
than the threshold. QUAL indicates the confidence
that the variant is genuine.
• 30—GATK
• 20—Starling
Part # 15028392 Rev. G
It is possible that some clusters will sequence beyond the sample DNA and read bases
from a sequencing adapter. This particularly applies to longer read lengths up to 250
cycles.
Using the Adapter setting in the Settings section of the sample sheet, you can specify the
5' portion of the adapter sequence to prevent reporting sequence beyond the sample DNA
in FASTQ files. Trimming the adapter sequence avoids reporting spurious mismatches
with the reference sequence, and improves performance in accuracy and speed of
alignment.
The Adapter setting performs adapter trimming or adapter masking depending on the
aligner used for the workflow. For workflows using BWA, reads are trimmed from the
start of the adapter sequence. When using Eland (deprecated in MiSeq Reporter v2.2),
reads are N-masked, or replaced with Ns (no-call), from the start of the adapter.
Read 1 and Read 2 Adapters
If you specify an adapter sequence using the Adapter setting in the sample sheet, the
same adapter is trimmed for Read 1 and Read 2. If you want to trim a different adapter
sequence in Read 2, use the AdapterRead2 setting in the sample sheet and specify the
adapter sequence in the same way that you would using the Adapter setting.
To trim two or more adapters, separate the sequences by a plus (+) sign.
How Adapter Trimming Works
MiSeq Reporter considers each potential adapter start position (n) within the sequence
starting at the first base (n=0). The process continues to count matches and mismatches
between sequence (n) and adapter (0), sequence (n + 1), and adapter (1), and so on. This
loop terminates if the following occurs:
MismatchCount > 1 and MismatchCount > MatchCount
Otherwise, the count continues until the end of the sequence or end of the adapter is
reached, whichever comes first. The sequence is trimmed starting at position n, if:
MatchCount / (MatchCount + MismatchCount) > Cutoff
By default, the cutoff is 0.9 or < 10% mismatch rate. This default setting can be modified
using the configurable setting AdapterTrimmingStringency.
Masking Short Reads
MiSeq Reporter includes a setting that prevents reads that have been almost entirely
trimmed or masked from confounding downstream analysis, which is based on the
following criteria:
} If the adapter is encountered within the first 32 bases of the read, the adapter
sequence is N-masked.
} If the adapter is identified in the first 32 bases and the read includes ten or more
bases from the start of the adapter, the entire read is N-masked.
This ten-base limit is controlled by the configuration setting NMaskShortAdapterReads.
15
Adapter Settings
Adapter Settings
Naming the Sample Sheet
During the run setup steps using MCS, the software automatically looks for a sample
sheet with a name that matches the barcode number of the reagent cartridge loaded on
the instrument. Therefore, Illumina recommends that you name your sample sheet with
the barcode number of the reagent cartridge that you will use for your run, followed by
*.csv extension. The barcode number is located on the reagent cartridge label directly
below the barcode.
In the following example, the sample sheet name is MS2000006-500.csv. You do not
need to include the kit version in the sample sheet name.
Figure 2 Reagent Cartridge Label
If you do not know which reagent cartridge you will use for your run, name your sample
sheet according to your preference followed by *.csv. When the software cannot locate
your sample sheet during the run setup steps, you can browse to the appropriate sample
sheet.
16
Part # 15028392 Rev. G
For technical assistance, contact Illumina Technical Support.
Table 1 Illumina General Contact Information
Illumina Website
Email
www.illumina.com
[email protected]
Table 2 Illumina Customer Support Telephone Numbers
Region
Contact Number
Region
North America
1.800.809.4566
Italy
Austria
0800.296575
Netherlands
Belgium
0800.81102
Norway
Denmark
80882346
Spain
Finland
0800.918363
Sweden
France
0800.911850
Switzerland
Germany
0800.180.8994
United Kingdom
Ireland
1.800.812949
Other countries
Contact Number
800.874909
0800.0223859
800.16836
900.812168
020790181
0800.563118
0800.917.0041
+44.1799.534000
MSDSs
Material safety data sheets (MSDSs) are available on the Illumina website at
www.illumina.com/msds.
Product Documentation
Additional product documentation in PDF is available for download from the Illumina
website. Go to www.illumina.com/support, select a product, then click Documentation &
Literature.
Illumina
San Diego, California 92122 U.S.A.
+1.800.809.ILMN (4566)
+1.858.202.4566 (outside North America)
[email protected]
www.illumina.com

MiSeq Sample Sheet Quick Reference Guide ®

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